Introduction

The liver is a vital organ responsible for protein metabolism, including synthesis and degradation. It produces nearly all blood proteins, except gamma globulins, and synthesizes a significant portion of amino acids. The liver plays a crucial role in the production of coagulation factors, such as fibrinogen and prothrombin, which are essential for blood clotting. Additionally, it contributes to red blood cell production by synthesizing erythropoietin, a hormone that regulates red blood cell formation. The liver also produces carrier proteins like albumin and transferrin, which transport hormones and nutrients. Its synthetic functions are indispensable for maintaining metabolic balance and overall health1.

Social isolation stress significantly increases the risk of liver disease. This highlights the intricate relationship between the social environment and liver health, emphasizing the importance of further research. Stress, both physiological and biological, is the organism’s response to stressors, including environmental conditions. Chronic stress can lead to oxidative stress and inflammation, which are key contributors to liver damage. Studies suggest that social isolation may exacerbate metabolic dysfunction and impair liver regeneration. Addressing social and environmental factors is crucial for mitigating stress-related liver disorders and improving overall health outcomes2,3.

The interactions between stress and liver function remain an area of ongoing research. However, it is well-established that stress negatively impacts the liver, impairing its activity and metabolic processes. Chronic stress can lead to oxidative stress and inflammation, which are significant contributors to liver damage. Studies suggest that stress-induced hormonal imbalances, particularly elevated cortisol levels, may exacerbate liver dysfunction. Additionally, stress has been linked to the progression of liver diseases such as non-alcoholic fatty liver disease (NAFLD) and fibrosis. Understanding these mechanisms is crucial for developing strategies to mitigate stress-related liver disorders and improve overall health outcomes4.

Stress affects the activity of the central nervous system and increases the secretion of hormones. The adrenal glands increase the secretion of the hormones epinephrine and norepinephrine by 10-fold or more. The action of these two hormones is to increase blood flow to the liver, resulting in the rapid breakdown of glycogen, proteins, and fats5. Stress can reduce growth performance, damage liver structure, and disrupt hepatic glucose metabolism and lipid metabolism6. Social isolation, characterized by a lack of social interaction and isolation from normal social environments, has emerged as a major concern in modern society, with profound implications for mental and physical health. Among the myriad consequences associated with social isolation, changes in liver function have received attention due to their potential implications for overall health7. Social isolation initiates oxidative stress and impairs hepatic insulin sensitivity. Social isolation induces hepatic hypertrophy in mice8. Oxidative stress is an imbalance between free radicals and antioxidants in the body. This means there are too many free radicals and not enough antioxidants. As a result, the excess free radicals begin to damage the body’s cells and tissues. Oxidative stress is a major cause of liver damage9. Conclusion.

N-acetylcysteine reduced the duration of violent behaviors, increased the duration of communication behaviors, reduced obsessive-compulsive behaviors, and reduced anxiety. This substance increased serum antioxidant capacity and increased liver catalase levels. N-acetylcysteine reduced serum malondialdehyde and reduced the expression of β IL-1 and IL-6 genes in the liver. Considering the above, it can be suggested that researchers study the use of this substance to reduce the effects of stress on the human body. Malondialdehyde is an organic compound with the nominal chemical formula CH2 (CHO) 2. A colorless liquid, malondialdehyde is a highly reactive compound that exists as an enol and is an indicator of oxidative stress. Increased free radicals cause excessive production of malondialdehyde10. Social isolation is a state of complete or near-complete lack of contact between an individual and society. This condition is distinct from loneliness, which refers to a temporary and involuntary lack of contact with other people in the world. Social isolation may be a problem for people of all ages, although symptoms are likely to vary by age group. Inflammation plays a central role in the pathogenesis of liver injury and disease, and social isolation has a role as a potential modulator of inflammatory processes in the liver11. Interleukins are cytokines produced by a variety of white blood cells that often affect other lymphocytes. These compounds play an important role in the immune system. Recent studies have revealed the involvement of cytokines of the interleukin-1 family in liver cell death. Upon activation of cell death induced by hepatic stimuli, IL-1 family cytokines released by dying liver cells stimulate the recruitment of immune cells, which in turn affect inflammation and subsequent liver damage. Thus, their potential as therapeutic targets in liver diseases is highlighted12. Interleukin 6 is one of the important interleukins in the body, secreted by white blood cells, and plays a role in inflammatory and immune responses. Interleukin 6 is secreted by macrophages, T-helper lymphocytes, B lymphocytes, and astrocytes, and is effective on plasma cells and lymphocytes. The main function of this cytokine is to promote faster differentiation of plasma cells and increased antibody production13. N-acetylcysteine is a drug used to treat paracetamol overdose and loosen thick mucus in people with chronic bronchopulmonary disorders such as pneumonia and bronchitis. N-acetylcysteine, a precursor of the endogenous antioxidant glutathione, is considered a promising therapeutic agent with diverse pharmacological properties, including anti-inflammatory and hepatoprotective effects14. N-acetylcysteine supplementation has been shown to reduce oxidative stress and inflammation. However, the potential protective effects of N-acetylcysteine specifically in the context of social isolation-induced liver dysfunction remain largely unknown15. This study was designed to investigate the effect of N-acetylcysteine ​​on the liver of offspring of mice subjected to social isolation stress. Understanding the impact of N-acetylcysteine on liver health in the context of social isolation stress is essential for the development of targeted interventions aimed at preserving liver function in vulnerable populations.

Results

Effect of N-acetylcysteine on the duration of communication behaviors in mice in the three-chamber sociability test

The results of the effect of N-acetylcysteine on the duration of communication behaviors in mice in the Three-chamber sociability test are shown in Fig. 1a. There was a significant difference in the duration of communication behaviors of mice between different groups. The duration of communication behaviors of mice in the social isolation stress group receiving normal saline was significantly reduced compared to the control group receiving normal saline. Also, the duration of communication behaviors of mice in the social isolation stress groups receiving N-acetylcysteine was significantly increased compared to the social isolation stress group.

Effect of N-acetylcysteine on the social preference time in mice in the three-chamber sociability test

The results of the effect of N-acetylcysteine on the social preference time in mice in the Three-chamber sociability test are shown in Fig. 1b. There was a significant difference in the social preference index time in mice between different groups. The social preference index time in mice in the socially isolated group receiving normal saline was significantly reduced compared to the control group receiving normal saline. Also, the social preference index time in mice in the socially isolated group receiving N-acetylcysteine ​​was significantly increased compared to the socially isolated group.

Fig. 1
figure 1

Effect of N-acetylcysteine on the duration of Sociability Index (SI) (a) and Social Novelty Preference Index (SNI) (b) in the Three-chamber sociability test, NS = normal saline, SIS = social isolation stress, NAC = N acetylcysteine, F = female, M = male, The results obtained from 8 mice are reported as Mean ± SD and analyzed by One-way ANOVA and Tukey’s post-test. ***p < 0.001 compared to the control group ###p < 0.001 compared to the SIS group receiving NS.

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Effect of N-acetylcysteine on the duration of violent behaviors in the resident-intruder test

The results of the effect of N-acetylcysteine on the duration of violent behaviors in the resident-intruder test are shown in Fig. 2. There was a significant difference in the duration of violent behaviors between the different groups. The duration of violent behaviors in the socially isolated group receiving normal saline was significantly increased compared to the control group receiving normal saline. Also, the duration of violent behaviors in the socially isolated group receiving N-acetylcysteine was significantly decreased compared to the socially isolated group.

Fig. 2
figure 2

Effect of N-acetylcysteine on the aggressive behaviors in Resident intruder test, NS = normal saline, SIS = social isolation stress, NAC = N acetylcysteine, F = female, M = male, The results obtained from 8 mice are reported as Mean ± SD and analyzed by One-way ANOVA and Tukey’s post-test. ***p < 0.001 compared to the control group, #p < 0.05, p < 0.01 compared to the SIS group receiving NS.

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Effect of N-acetylcysteine on serum antioxidant capacity

Serum antioxidant capacity in the socially isolated stress group receiving normal saline was significantly reduced compared to the control group receiving normal saline. Serum antioxidant capacity in the groups receiving N-acetylcysteine was significantly increased compared to the socially isolated stress group receiving normal saline (Fig. 3a).

Effect of N-acetylcysteine on serum malondialdehyde levels

The serum malondialdehyde levels in the socially isolated stress group receiving normal saline were significantly increased compared to the control group receiving normal saline. The serum malondialdehyde levels in the N-acetylcysteine group were significantly decreased compared to the socially isolated stress group receiving normal saline (Fig. 3b).

Fig. 3
figure 3

Effect of N-acetylcysteine on serum antioxidant capacity (a), serum malondialdehyde (b) NS = normal saline, SIS = social isolation stress, NAC = N acetylcysteine, F = female, M = male, The results obtained from 8 mice are reported as Mean ± SD and analyzed by One-way ANOVA and Tukey’s post-test.**p < 0.01, ***p < 0.001 compared to the control group #p < 0.05 compared to the SIS group receiving NS.

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Effect of N-acetylcysteine on catalase enzyme activity in liver tissue

Figure 4 shows that catalase enzyme activity in the group subjected to social isolation stress receiving normal saline was significantly decreased compared to the control group receiving normal saline. Catalase enzyme activity in liver tissue in the groups receiving N-acetylcysteine increased compared to the group subjected to social isolation stress receiving normal saline.

Fig. 4
figure 4

Effect of N-acetylcysteine liver catalase enzyme NS = normal saline, SIS = social isolation stress, NAC = N acetylcysteine, F = female, M = male, CAT = catalase, u/mg = unit per milligram, The results obtained from 8 mice are reported as Mean ± SD and analyzed by One-way ANOVA and Tukey’s post-test. ***p < 0.001 compared to the control group ##p < 0.01, ###p < 0.001 compared to the SIS group receiving NS.

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IL-1β gene expression level

There was a significant difference in IL-1β gene expression level between different groups. IL-1β gene expression level in the group subjected to social isolation stress receiving normal saline was significantly increased compared to the control group receiving normal saline. IL-1β gene expression level in the group subjected to social isolation stress receiving N-acetylcysteine was significantly decreased compared to the group subjected to social isolation stress (Fig. 5a).

IL-6 gene expression levels

The IL-6 gene expression levels in the socially isolated stress group receiving normal saline were significantly increased compared to the control group receiving normal saline. The IL-6 gene expression levels in the socially isolated stress group receiving N-acetylcysteine were significantly decreased compared to the socially isolated stress group (Fig. 5b).

Fig. 5
figure 5

Effect of N-acetylcysteine on gene expression levels of interleukin 1 beta (a) and interleukin 6 (b), NS = normal saline, SIS = social isolation stress, NAC = N acetylcysteine, F = female, M = male, The results obtained from 8 mice are reported as Mean ± SD and analyzed by One-way ANOVA and Tukey’s post-test. **p < 0.01, ***p < 0.001 compared to the control group ##p < 0.01, ###p < 0.001 compared to the SIS group receiving NS.

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Discussion

The aim of this study was to investigate the effects of N-acetylcysteine on the liver of socially isolated stressed mice with the aim of evaluating behavioral and biochemical changes. The results obtained are based on behavioral, biochemical, and gene expression analyses. Stress is the body’s natural response to stimuli and internal or external pressures.

Stress responses vary widely among individuals due to a combination of genetic, psychological, and environmental factors. Some people may experience heightened physiological reactions, while others may show resilience and adapt more effectively. Factors such as social support, coping mechanisms, and past experiences play significant roles in determining how one reacts to stress. Understanding these differences is crucial for developing personalized stress management strategies16. Stress is a mental and physical reaction to life experiences that can seriously damage a person’s health. When a person is stressed, natural killer cells in the liver increase, and some cases, cause liver cell death and worsen liver disease17. Social isolation, characterized by a lack of social interaction and deprivation from normal social environments, has emerged as a major concern in modern society. Among the myriad consequences associated with social isolation, changes in liver function have attracted much attention18. Antioxidants are chemicals that appear to reduce the effects of oxidative stress on the body. Oxidative stress is thought to cause liver disease. Reduced antioxidant activity has been implicated in the etiology of the liver disease. Evidence suggests that antioxidant levels in the blood and brain of individuals under stress are altered several studies have also shown that an imbalance between overproduction of reactive oxygen species and antioxidant capacity plays a role in susceptible individuals. Tissue damage caused by oxidative stress plays a major role in the pathogenesis of social isolation stress19,20. Since N-acetylcysteine is a potent antioxidant, the present study investigated the effect of N-acetylcysteine on the liver of socially isolated mice. Extensive evidence suggests that the proven therapeutic properties of N-acetylcysteine, through increasing endogenous antioxidants such as glutathione to prevent oxidative stress and reduce inflammation, are essential for improving liver function in patients undergoing liver surgery21.

In the present study, anxiety-like behaviors, deficits in social interactions, and liver tissue abnormalities were observed in the socially isolated stress group receiving normal saline in the social interaction test and the resident intruder test. Previous studies have also shown that behavioral changes caused by social isolation stress at birth can be observed2,3. In the present study, it was observed that in the sociability test, the duration of communication behaviors of mice in the group subjected to social isolation stress receiving normal saline was significantly reduced, and the administration of N-acetylcysteine significantly increased the duration of communication behaviors of mice. This is a good indication of the anti-anxiety effects of N-acetylcysteine. In line with the present findings, Wei et al. (2020) stated that maternal separation stress in the Three-chamber sociability test caused impairment in social cognition or repetitive behavior22. The results of the assessment of violent behaviors in the resident intruder test showed that social isolation stress significantly increased the duration of violent behaviors in mice. Administration of N-acetylcysteine significantly reduced the duration of violent behaviors in the resident intruder test. Our results are consistent with the study by Shin et al. in 2019. Oxidative stress is one of the mechanisms associated with social isolation stress. Varying degrees of oxidative damage and reduction of antioxidant enzymes have been reported in people with anxiety. Oxidative stress occurs when the body’s antioxidant defenses are insufficient to combat free radicals. Increased free radicals in oxidative stress cause damage to the nervous system in various ways and cause complications such as depression, diabetes, and Parkinson’s23,24,25,26. Social stress is associated with reduced antioxidant levels and induction of oxidative activity27. The results of the present study showed that serum antioxidant capacity was significantly reduced in the group subjected to social isolation stress receiving normal saline compared to the control group. Treatment of the groups with N-acetylcysteine increased serum antioxidant capacity compared to the group under social isolation stress. Some studies have shown that serum total antioxidant capacity is reduced in people with major depression compared to normal people28,29,30,31. In a model of chronic stress caused by social isolation, depressive-like behaviors have been observed along with increased corticosterone levels, decreased adrenocorticotropic hormone, and decreased serum antioxidant capacity32. Erdogan et al. showed in a study that N-acetylcysteine supplementation reduces oxidative stress and inflammation and improves liver function in various experimental models of liver injury and disease. However, the potential protective effects of N-acetylcysteine specifically in the context of social isolation stress-induced liver dysfunction remain largely unknown33. N-acetylcysteine induces modulating effects on hepatotoxicity by inhibiting free radical production and increasing antioxidant capacity28,29,30. N-acetylcysteine administration can reduce oxidative stress and improve liver histology in mice with nonalcoholic steatohepatitis31,34. In the present study, serum malondialdehyde was significantly increased in the social isolation stress group receiving normal saline compared to the control group. Treatment of the social isolation stress group with N-acetylcysteine reduced serum malondialdehyde levels compared to the social isolation stress group that received normal saline. The results of a 2020 study by Kang et al. showed that N-acetylcysteine reduced oxidative stress, liver damage, and intestinal tissue damage. Researchers have shown that N-acetylcysteine has therapeutic applications in livestock to modulate immune responses, liver damage, and oxidative stress35. Catalase is a major component of the antioxidant defense system that reduces hydrogen peroxide through water-reducing mechanisms. Catalase is more active at higher concentrations of hydrogen peroxide. Malcon et al. found that animals with multiple sclerosis had significantly lower levels of catalase in the hippocampus, but no changes were observed in plasma. This prevents oxidative damage to the brain36. In the present study, liver catalase was significantly decreased in the social isolation stress group receiving normal saline compared to the control group. Liver catalase levels increased in the social isolation stress group receiving N-acetylcysteine compared to the social isolation stress group receiving normal saline. Studies on disorders caused by social isolation indicate the prominent role of inflammation and the innate immune system in the occurrence of diseases. Inflammasomes are intracellular multi-protein complexes whose accumulation causes the production of pro-inflammatory cytokines such as IL-β1 and IL-6. Interleukin-1 beta (IL-1β) is a pro-inflammatory cytokine involved in immune responses, inflammation, and cell death. It plays a key role in conditions like asthma and autoimmune diseases. Interleukin-6 (IL-6) is another cytokine that regulates immune responses and inflammation, and is linked to diseases such as rheumatoid arthritis, diabetes, and severe COVID-194. Both cytokines are crucial in understanding and managing inflammatory and autoimmune conditions37,38. Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), and Tumor Necrosis Factor-alpha (TNF-α) are key pro-inflammatory cytokines involved in immune responses and inflammation. NF-κB is a transcription factor that regulates the expression of these cytokines and plays a crucial role in inflammatory responses. Social isolation stress can trigger these inflammatory pathways, leading to increased levels of IL-1β, IL-6, and TNF-α. N-acetylcysteine (NAC) has antioxidant and anti-inflammatory properties that may help mitigate these effects by reducing oxidative stress and inflammation39,40,41,42. The results of this study on IL-1β gene expression showed that treatment with N-acetylcysteine led to a decrease in the expression level of this gene. In our study, environmental inflammatory factors including IL-1β were significantly increased in mice subjected to social isolation stress, which is in line with previous studies that indicated that people who experience adversity early in their lives have a significant increase in CRP and IL-6 levels compared to healthy individuals43. Treatment with N-acetylcysteine significantly reduced IL-1β. N-acetylcysteine (NAC) shows promise in treating stress-related conditions like anxiety, depression, and PTSD by reducing oxidative stress and inflammation. It also has potential benefits in managing non-acetaminophen-induced acute liver failure by improving liver function and reducing oxidative damage. Further research is needed to confirm its efficacy and optimal usage in these conditions44,45,46. In this study, no significant differences were observed between male and female subjects in any of the test results. Given that this study examined the short-term effects of N-acetylcysteine, it may be possible to gain a deeper understanding of its effects through long-term studies on this substance. It is also necessary to design and implement future studies, taking into account ethical issues on human society, to better understand the effects of this substance on humans. These additional studies can address many issues in this regard.

Conclusion

N-acetylcysteine reduced the duration of violent behavior, increased the duration of communicative behavior, reduced obsessive behavior, and alleviated anxiety. This substance increased serum antioxidant capacity and liver catalase levels. N-acetylcysteine also reduced serum malondialdehyde and the expression of IL-1β and IL-6 genes in the liver. Considering these findings and its potential use in humans, it is necessary for researchers to conduct broader studies on this substance to reduce the effects of stress on the body. Such studies should observe necessary ethical considerations.

Materials and methods

Laboratory animals

After obtaining the code of ethics, ten male and ten female laboratory mice were purchased from the Pasteur Institute of Tehran. Male and female mice were placed one by one in separate cages and mated. The pups were fed with breast milk and standard food for three weeks with their parents. At the end of the third week, the groups that were to be socially isolated according to Fig. 6 were separated from their parents and placed alone in Plexiglas cages for four weeks. The control group remained with their parents for this four-week period. All groups were maintained under standard laboratory conditions (12-hour light/dark cycle, temperature 22 ± 1 °C, and free access to food and water). The mice were 8 to 12 weeks old and weighed 25 to 30 g at the time of the experiments.

The pups were divided into 6 groups as follows:

  1. 1.

    Eight female pups received normal saline (10 mg/kg) as a control group.

  2. 2.

    Eight female pups were subjected to social isolation stress and received normal saline.

  3. 3.

    Eight female pups were subjected to social isolation stress and received N-acetylcysteine ​​(150 mg/kg).

  4. 4.

    Eight male pups received normal saline as a control group.

  5. 5.

    Eight male pups were subjected to social isolation stress and received normal saline.

  6. 6.

    Eight male pups were subjected to social isolation stress and received N-acetylcysteine (150 mg/kg).

Behavioral tests were performed on these mice at puberty. After anesthesia with ketamine and xylazine at doses of 100 and 10 mg/kg, blood samples were taken. They were then killed by decapitation and liver tissue was harvested47 (Fig. 6).

Fig. 6
figure 6

Schematic diagram of the study design. NS = normal saline, SIS = social isolation stress, NAC = N-acetylcysteine, n = number.

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.

Social isolation stress model

Mice were housed individually in cages from the end of the third week of life for 4 weeks. The cages of these mice were covered with black paper except for the lid, and to minimize contact, the mice were only taken out of the cage once a week by one person to clean the cage48,49.

N-acetylcysteine treatment

N-acetylcysteine was prepared in 0.9% normal saline and injected intraperitoneally into mice at a dose of 150 mg/kg daily for twenty-eight days during the induction of the social isolation stress model50,51,52.

Behavioral tests

Three-chamber sociability test

Thirty minutes before the behavioral tests, the cages of the mice were moved to a special room. A large polycarbonate box was used to conduct this test, which was divided into three compartments by two movable walls. To begin with, the chambers were separated by placing two walls, and two small wire cages were placed in each of the right and left chambers. The test mouse was placed in the middle cage for five minutes to acclimate. New mouse number 1, which was similar in breed, color, and weight to the test mice, was placed in one of the wire cages. Then, the wall between the middle chamber and the two side chambers was removed, and for ten minutes, the duration and number of times the test mouse interacted with new mouse 1 in the cage were calculated and recorded by camera. At this stage of the experiment, normal mice usually spend most of their time in the chamber containing a novel animal, rather than in the chamber containing an empty box, as they try to identify this novel animal well. After ten minutes, new mouse number 2 was placed in the other chamber, and the partition between the middle chamber and the two side chambers was removed. At this stage, when normal mice see two mice in the two side chambers, they spend more time with the novel mouse than with the older mouse. They try not to stay too long with the mouse they have already examined and evaluated, spending most of their time getting to know the second mouse, which is newer. All the observations mentioned for the first novel mouse were also measured for the second mouse for ten minutes. The numerical values of SI and SNI were calculated using the following formulas53.

$$\:\text{S}\text{o}\text{c}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\text{I}\text{n}\text{d}\text{e}\text{x}\:\left(\text{S}\text{I}\right)=\frac{\text{t}\text{i}\text{m}\text{e}\:\text{e}\text{x}\text{p}\text{l}\text{o}\text{r}\text{i}\text{n}\text{g}\:\text{n}\text{o}\text{v}\text{e}\text{l}\:\text{m}\text{o}\text{u}\text{s}\text{e}\:1-\:\text{t}\text{i}\text{m}\text{e}\:\text{e}\text{x}\text{p}\text{l}\text{o}\text{r}\text{i}\text{n}\text{g}\:\text{n}\text{o}\text{v}\text{e}\text{l}\:\text{o}\text{b}\text{j}\text{e}\text{c}\text{t}}{\text{t}\text{i}\text{m}\text{e}\:\text{e}\text{x}\text{p}\text{l}\text{o}\text{r}\text{i}\text{n}\text{g}\:\text{n}\text{o}\text{v}\text{e}\text{l}\:\text{m}\text{o}\text{u}\text{s}\text{e}\:1+\:\text{t}\text{i}\text{m}\text{e}\:\text{e}\text{x}\text{p}\text{l}\text{o}\text{r}\text{i}\text{n}\text{g}\:\text{n}\text{o}\text{v}\text{e}\text{l}\:\text{o}\text{b}\text{j}\text{e}\text{c}\text{t}}$$
$$\:\text{S}\text{o}\text{c}\text{i}\text{a}\text{l}\:\text{N}\text{o}\text{v}\text{e}\text{l}\text{t}\text{y}\:\text{P}\text{r}\text{e}\text{f}\text{e}\text{r}\text{e}\text{n}\text{c}\text{e}\:\text{I}\text{n}\text{d}\text{e}\text{x}\:\left(\text{S}\text{N}\text{I}\right)=\frac{\text{t}\text{i}\text{m}\text{e}\:\text{e}\text{x}\text{p}\text{l}\text{o}\text{r}\text{i}\text{n}\text{g}\:\text{n}\text{o}\text{v}\text{e}\text{l}\:\text{m}\text{o}\text{u}\text{s}\text{e}\:2-\:\text{t}\text{i}\text{m}\text{e}\:\text{e}\text{x}\text{p}\text{l}\text{o}\text{r}\text{i}\text{n}\text{g}\:\text{k}\text{n}\text{o}\text{w}\text{n}\:\text{m}\text{o}\text{u}\text{s}\text{e}}{\text{t}\text{i}\text{m}\text{e}\:\text{e}\text{x}\text{p}\text{l}\text{o}\text{r}\text{i}\text{n}\text{g}\:\text{n}\text{o}\text{v}\text{e}\text{l}\:\text{m}\text{o}\text{u}\text{s}\text{e}\:2-\:\text{t}\text{i}\text{m}\text{e}\:\text{e}\text{x}\text{p}\text{l}\text{o}\text{r}\text{i}\text{n}\text{g}\:\text{k}\text{n}\text{o}\text{w}\text{n}\:\text{m}\text{o}\text{u}\text{s}\text{e}}$$

Resident-intruder paradigm

In this experiment, mice that had been socially isolated, each living alone in its own cage for four weeks, were brought into the testing room one by one. These mice are known as resident mice. Then, a mouse of the same breed, sex, and color, but with slightly lower weight, was introduced as an intruder mouse into the cage of the resident mice. For 10 min, the violent and aggressive attacks of the resident mouse towards the intruder mouse were recorded and counted by a camera. The mice in the control group were also brought into the testing room with the same territory, and the bedding inside the cage was not changed for at least a week. In this case, only the mouse that was to be tested as a resident remained in the cage for the experiment, and the other mice were moved to another cage. The experiment was conducted on this mouse only in its own cage as a resident mouse, and the intruder mouse entered its cage as mentioned above. Normal mice in this experiment usually showed less violent movements and were more likely to interact with the newcomer, the intruder54,55.

Gene expression

Tissue samples will be prepared from the liver of mice. Finally, the expression levels of IL-1beta and IL-6 genes in the liver were measured by RT-PCR56.

Quantitative real-time polymerase chain reaction (qRT-PCR)

After complete anesthesia, the liver was removed from the body and immediately frozen in liquid nitrogen and stored at -80 °C until analysis. RNA from liver tissue was extracted using Trizol reagent (Invitrogen, Cergy Pontoise, France). Changes in mRNA levels were assessed by qRT-PCR57. Specifically, 1 µg of RNA from each sample underwent reverse transcription with the Prime Script RT reagent kit (Takara Bio Inc., Otsu, Japan). qRT-PCR was conducted using SYBR Premix Ex Taq technology (Takara Bio) on a Roche photocycle (Roche Diagnostics, Mannheim, Germany). Primer sequences are listed in Table 1. B2m served as the housekeeping gene, and fold changes in the expression of each target mRNA relative to B2m were calculated using the 2-ΔΔCt formula. (Table 1)

Table 1 The genes and primer sequences used in polymerase chain reaction (PCR) amplification.
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Biochemical tests

Determination of serum antioxidant capacity

To measure antioxidant capacity using the Ferric Reducing Power method, three solutions were used, including solution No. 1: buffer (1.55 mL sodium acetate and 8 mL concentrated acetic acid, made up to 500 mL with distilled water), solution No. 2 ferric chloride solution (270 mg FeCl3 (H2O) 6, made up to 50 mL with distilled water), and solution No. 3 triazine solution (47 mg triazine dissolved in 40 mL 40 mM HCl). The working solution was prepared by adding 10 mL of solution No. 1, 1 mL of solution No. 2, and 1 mL of solution No. 3. 25 µl of serum or tissue homogenate sample was added to 1.5 ml of working solution and incubated at 37 °C for 10 min, then the optical absorption was recorded at a wavelength of 593. Using a standard curve, the antioxidant capacity of the samples was recorded58.

Determination of liver malondialdehyde

One ml of liver homogenate solution was mixed with trichloroacetic acid (TCA) thiobarbituric acid (TBA) and HCL with pH = 1.6–1.7 and placed at 95 °C for 45 min. After cooling the samples for 1 h, 4 ml of n-butanol was added and centrifuged at 1000 rpm for 10 min. After that, the absorbance of the supernatant was read at 532 nm using a plate reader.

Then, the malondialdehyde concentration was calculated in nanomoles per gram using the following formula59.

MDA concentration = absorbance/1.56 × 105.

Determination of liver tissue catalase

To measure tissue catalase activity in mice, researchers often use spectrophotometric methods due to their precision and reliability. Here’s a general overview based on recent scientific advancements:

Sample preparation: Tissue samples, such as liver or kidney, are homogenized in a suitable buffer (e.g., phosphate buffer) to extract catalase. The homogenate is then centrifuged to remove debris, and the supernatant is collected for analysis.

Reaction setup: The assay typically involves incubating the tissue extract with hydrogen peroxide (H₂O₂). Catalase breaks down H₂O₂ into water and oxygen, and the rate of this reaction is measured.

Detection

Spectrophotometric assay: The decrease in H2O2 concentration is monitored by measuring the absorbance at 240 nm60.

Data analysis

Graph Pad Prism software (Version 8) was used for data analysis. Kolmogorov–Smirnov test was applied to evaluate the normal distribution of data. Using the Brown-Forsythe test, the homogeneity of variances has been checked. Statistical data analysis was conducted through a one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Results were deemed statistically significant at p < 0.05.